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Biological and Medical Applications of Materials and Interfaces

Enhanced Metabolic Activity of Cytochrome P450 via Carbon Nanocages Based Photochemical Bionanoreactor Ling Jiang, Kan Wang, Fen Zhang, Yuanjian Zhang, Huaisheng Wang, and Songqin Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b14810 • Publication Date (Web): 13 Nov 2018 Downloaded from http://pubs.acs.org on November 14, 2018

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ACS Applied Materials & Interfaces

Enhanced Metabolic Activity of Cytochrome P450 via Carbon Nanocages Based Photochemical Bionanoreactor Ling Jianga, Kan Wanga, Fen Zhanga, Yuanjian Zhanga, Huaisheng Wangb,*, Songqin Liua,*

aKey

Laboratory of Environmental Medicine Engineering, Ministry of Education, Jiangsu

Engineering Laboratory of Smart Carbon-Rich Materials and Device, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 210096, PR China bDepartment

of Chemistry, Liaocheng University, Liaocheng, Shandong, 252059, China

Corresponding

author: [email protected]; [email protected]. Fax: 86-25-52090613, Tel: 86-25-52090613.

ACS Paragon Plus Environment

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ABSTRACT: Recently, the early screening of the genotoxicity of new chemicals and drugs calls for the envelope of micro/nanoreactors for metabolic study. Herein, a novel light-driven enzymatic bionanoreactor is designed with the gold nanoparticles modified carbon

nanocage

(Au@CNC)

as

nanoreactor,

and

meso-tetrakis(4-carboxyphenyl)porphyrin (TCPP) as photosensitizer for cytochrome P450-mediated drug metabolism. By confining cytochrome P450 3A4 (CYP3A4) enzyme and TCPP inside pores of Au@CNC, a high metabolic activity is achieved by using 7-ethoxytrifluoromethyl coumarin (7-EFC) as substrate due to the 3D hierarchical porous structure, large surface area, and fast electron transfer capacity of Au@CNC. It is noted that owing to the presence of Au NPs inside CNC, the surface hydrophilicity of CNC is much improved, which further promote the catalytic activity of CYP3A4 enzyme. To our knowledge, this is the first attempt to apply CNC as a bionanoreactor for NADPH-free and light-driven in vitro drug metabolism. In addition, the presented bionanoreactor exhibits a variety of advantages in terms of fast response, short assay time (10 min), high sensitivity and good selectivity, which is expected to expedite the drug screening and render potential advances in drug discovery and development. KEYWORDS: Cytochrome P450, carbon nanocage, light-driven bio-nanoreactor, drug metabolism


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1. INTRODUCTION Cytochrome P450 enzymes (CYP450) are heme-containing transmembrane proteins, anchored to the membrane by the N-terminal hydrophobic helix,1-3 which metabolize a plenty of endogenous and xenobiotic molecules.4,5 The transmembrane region of microsomal CYP450 is indispensable for their functions due to only 40% of enzymatic activities are reserved when lack of this membrane anchor.6 As the most highly expressed xenobiotic enzymes in the liver, they undertook a wide variety of phase I reactions, metabolizing approximately 70%~80% of all clinical used drugs.7 Particularly, cytochrome P450 3A4 (CYP3A4), the most richly expressed isoform in the liver, is able to interact with numerous structurally diverse compounds and then metabolize such molecules and drugs.8,9 As is well known, two electrons are required for the CYP450 enzyme induced metabolic reaction.10 Great efforts have been devoted by using chemical and electrochemical methods to provide two electrons to trigger the P450-mediated reactions.11-15 Among these, the light-driven method for P450-mediated pharmaceutical metabolism has attracted extensive attention in the last few years by utilizing diverse photosensitizer such as quantum dots and eosin Y as electron donors.16-19 These light-driven methods exhibited good selectivity and sensitivity between the used CYP450 enzymes and their substrates, showing great potential for in vitro P450-mediated pharmaceutical metabolism investigation. To improve the kinetics of enzymatic reaction, numbers of micro/nanoreactors have



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been developed by confining enzymes spatially in a small space like anchor active enzymes on the cell membrane or fixed in a specific region of the cell. A series of smart nanostructured materials, such as porous alumina,20-22 TiO2 nanotubes arrays,23,24 nanoporous graphene foams,25 mesoporous silica26,27 and macroporous ordered silica foam (MOSF),28-31 have been explored to construct enzymatic micro/nanoreactors. These micro/nanoreactors exhibited distinctive advantages, such as fast reaction kinetics, good stability and enhanced catalytic activity. Since both enzymes and substrates are enriched in the pores/channels of these materials, the mass-transfer is benefited by the delivery of the substrate from the solution to the active centers of enzymes and the diffusion of the metabolite from the channels/pores to the solution, which elevated the catalytic efficiency. For example, based on eosin-Y modified MOSF, Liu et al. developed an efficient photochemical bionanoreactor for P450-catalyzed pharmaceutical metabolism with the assistance of the irradiation of green light.30 Also, in our previous work, by using MOSF as a macroreactor and the phthalocyanine cobalt as a photosensitizer, a photo-driven enzymatic reaction system was constructed, and both the enzymatic kinetics and metabolic efficiency were immensely enhanced via restricting enzyme/photosensitizer in MOSF with suitable pore environment.31 Inspired by the powerfully application of confining enzyme/photosensitizer in nanostructured materials for drug metabolism, herein, a novel light–driven enzymatic bionanoreactor was fabricated by using gold nanoparticles modified 3D hierarchical carbon-based

nanocages

(Au@CNC)

as

nanoreactor


and

meso-tetrakis(4-

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carboxyphenyl)porphyrin (TCPP) as photosensitizer. By confining CYP3A4 enzyme and TCPP in the pores of Au@CNC, the enhanced metabolic activity and fast reaction kinetics were realized when using 7-ethoxytrifluoromethyl coumarin as substrate. This could be attributed to the excellent conductivity, good biocompatibility, large surface area and good surface hydrophilicity of Au@CNC, which provided the suitable microenvironment to maintain the stability and catalytic activity of CYP3A4. 2. EXPERIMENTAL SECTION 2.1. Chemicals. Cytochrome P450 3A4 recombinant human isozyme protein (CYP3A4, 1 nmol, 0.5 mL) contained cytochrome P450-reductase (CPR) in 0.1 M potassium phosphate buffer (pH 7.4) was expressed in baculovirus-infected insect cells (BTI-TN-5B1-4) and purchased from Chaoyan Biotechnology Co. Ltd. (Shanghai, China). The CYP3A4 enzyme has a full length wild-type sequence (see Supporting Information). Chloroauric acid (HAuCl4∙4H2O), cysteamine, meso-tetrakis(4-carboxyphenyl)porphyrin (TCPP), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), sodium borohydride (NaBH4), N-hydroxysuccinimide (NHS), 7-ethoxytrifluoromethyl coumarin (7-EFC), ketoconazole (≥95%) and resorufin ethyl ether (7-ER) were received from Sigma-Aldrich (Shanghai, China). Phosphate buﬀer solution (PB) was obtained by blending 0.1 M NaH2PO4 and Na2HPO4. Deionized water was used throughout the experiment. 2.2. Instruments. X-ray photoelectron spectroscopy (XPS) was operated on a



multitechnique surface analysis system (Thermo Electron Co., USA). The specific surface areas of samples were measured by a Thermo Fisher Scientific Surfer Gas Adsorption Porosimeter, and before the measurements, all the samples were degassed at 200 °C for 6 h. The transmissio elenctron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM) images was collected by transmission electron microscope (JEM−2010, JEOL, Japan). An Ultra Plus field emission scanning electron microscope (SEM, Zeiss, Germany) was used to observe the surface morphological features of the carbon-based nanomaterials. UV-vis absorption spectra were recorded with Cary 100 (Agilent, Singapore). The fluorescence (FL) spectra were obtained from a FluoroMax-4 spectrometer (Horiba, Japan). Zeta potential data were measured by a zeta potential analyzer (Omni, Brookhaven, USA) at 25 °C. Static water contact angles were determined by an OCA 15Pro contact angle meter at 25 °C, and the volume of each water droplet was 5 μL. 2.3. Preparation of enzymatic reactor. The CNC nanomaterial was prepared on the basis of MgO template with benzene as precursor at 850 °C.32 Gold nanoparticles (Au NPs) embedded CNC nanomaterial was synthesized as follows: 1.0 mg of CNC was dispersed in 5 mL water. Afterwards, 20 μL of HAuCl4 (10 mM) solution was dropped in the suspension and stirred for 12 h, making the Au3+ contact adequately with the negatively charged pores of CNC through electrostatic incorporation.33 Then, 60 μL of NaBH4 (0.5 mg mL−1) solution was rapidly added and reacted for 30 min. The obtained mixture was centrifuged and washed repeatedly. Finally, the obtained product was


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dispersed into 2 mL water and denoted as Au@CNC. Subsequently, 0.5 mL of cysteamine (25 mg mL−1) was added into the Au@CNC dispersion and the mixture was stirred for 2 h. Then the mixture was centrifuged at 12,000 rpm and washed with water repeatedly to obtain the cysteamine modified Au@CNC. Afterwards, 2.0 mg of TCPP was added into the above suspension and reacted for another 0.5 h with the activation of excess EDC and NHS. Finally, the resulting suspension was centrifuged at 8000 rpm and washed with water for three times to remove the unbound TCPP. The conjugation of CYP3A4 and TCPP/Au@CNC was performed by the carbodiimide coupling between the amine groups of CYP3A4 and carboxyl groups of TCPP.34 Briefly, 10 μL aqueous stock solution of CYP3A4 was added to 50 μL of TCPP/Au@CNC and incubated for 0.5 h. Then the conjugate of CYP3A4/TCPP/Au@CNC was obtained by centrifugation and re-dispersed in PB buffer (0.5 mL). The process was depicted in Scheme 1. 2.4. Light-driven bioconversion of 7-EFC by CYP3A4/TCPP/Au@CNC. 10 μL of 7-EFC and 50 μL of CYP3A4/TCPP/Au@CNC were added to 2.0 mL 0.1 M pH 7.4 PB containing 1 mM ascorbic acid (AA). AA worked as an electron supplier to fulfill the reaction cycle. The mixture was irradiated under visible light for 10 min with mild stirring, and the conversion of 7-EFC was monitored by fluorescence spectroscopy. Meanwhile, the metabolic products were further confirmed by HPLC analysis, and the detailed detection process was described in the Supporting Information.



3. RESULTS AND DISCUSSION 3.1. Characterization of the photochemical bionanoreactor. The CNC was firstly modified with AuNPs via an in-situ chemical reaction. The growing amount of AuNPs and the property of the resulting Au@CNC were optimized by varying the concentration of the precursors (HAuCl4) (Figure S1). The XPS spectrum of Au@CNC composite distinctly manifested the existence of Au 4f, C 1s and O 1s peaks, while CNC sample was only composed of C 1s and O 1s two peaks (Figure S2A and B). And the contact angle of Au@CNC hybrid changed to 68°, demonstrating that the surface wettability of Au@CNC was much enhanced compared to that of pristine CNC nanomaterial (135°), owing to the change of the surface chemistry and the decrease of the total surface roughness after attaching AuNPs inside the pores of CNC,35 which was favorable for loading more enzymes/drugs and accelerating the transport of mass and electrons,36 and thus finally improving the efficiency of the enzymatic biocatalytic reaction. Meanwhile, the SEM and TEM images showed that both CNC and Au@CNC exhibited 3D hierarchical porous structures (Figure 1), indicating the structure of CNC was well remained after the in-situ deposition of Au NPs. The shell thickness of CNC was about 3.5 nm (inset of Figure 1C), which was beneficial for mass-transfer between the inside and outside of the nanocages,37,38 i.e., the delivery of substrate from solution to the CYP3A4 encapsulated in the pores of Au@CNC, and the diffusion of the metabolite from the pores of Au@CNC to the solution. Additionally, comparing to pristine CNC, AuNPs (~5 nm) were well embedded in the pores of CNC, without any apparent agglomeration (Figure


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1D). The specific surface area of Au@CNC was 1094.1 m2 g−1, smaller than pristine CNC (1778.5 m2 g−1), and the pore volumes of CNC and Au@CNC were 3.32 and 2.01 m3 g−1 (Figure S1C and D), respectively. These results confirmed the effective modification of Au NPs inside the pores of CNC. The successful assembly of the photochemical bionanoreactor was verified by UV-vis absorption spectra. No characteristic peak of AuNPs for Au@CNC at ~510 nm was observed owing to the small size of AuNPs and most of them attached inside the pores of CNC (Figure 2A). However, TCPP/Au@CNC displayed a strong Soret absorption at 413 nm along with four Q bands at 514, 549, 589, and 645 nm, referable to the −* transition between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of TCPP (Figure S3).39 After CYP3A4 coupled with carboxyl groups of TCPP, there was a new peak appeared at 260 nm for CYP3A4/TCPP/Au@CNC, which suggested the successful conjugation of CYP3A4 to TCPP/Au@CNC. The assembly of CYP3A4/TCPP/Au@CNC was also demonstrated by zeta potential (ξ) measurements (Figure 2B). The original CNC had a ξ of –28.4 mV and changed to –22.8 mV after absorption of Au3+, indicating Au3+ was well absorbed into the pores of CNC through electrostatic interaction. The in-situ reduction of AuNPs led the ξ value of Au@CNC slightly shifted to –26.9 mV.40 After negatively charged TCPP coupling, the ξ of TCPP/Au@CNC was shifted to –37.6 mV. When CYP3A4 further binded to TCPP/Au@CNC, the ξ value of the resulting CYP3A4/TCPP/Au@CNC changed to –13.5 mV due to the conjugation of positively charged CYP3A4.33



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3.2. Photoelectrochemical property of CYP3A4/TCPP/Au@CNC. The bionanoreactor was fabricated by using TCPP as photosensitizer and Au@CNC as nanoreactor, where CYP3A4 was coupled with TCPP and confined in pores of Au@CNC. Under the visible light, the electron–hole pairs were generated by transferring electrons from the HOMO to the LUMO of TCPP, and then the light-induced electrons in TCPP were injected to the heme active sites of CYP3A4 for driving the catalytic reaction. To investigate the feasibility of the cytochrome P450-catalyzed enzymatic reaction by TCPP/Au@CNC under the light irradiation, the photo-induced current was tested with a conventional electrochemical system (Figure 2A). When TCPP was coated onto ITO electrode, a distinct cathodic photocurrent of TCPP/ITO (curve b) was emerged compared to that of bare ITO (curve a), which could be ascribed to the generation of the electron–hole pair and the electron transfer between TCPP and the ITO electrode. However, the photocurrent of TCPP/Au@CNC was immensely increased (curve c), owing to the excellent

conductivity

of

Au@CNC.

After

CYP3A4

covalently

linked

to

TCPP/Au@CNC, the photocurrent of CYP3A4/TCPP/Au@CNC/ITO further increased (curve d). As known, the LUMO and HOMO positions of TCPP were located at –3.60 and –5.60 eV,36 respectively, while the energy level of CYP3A4 was –4.26 eV.31,42 As a result, the redox energy of CYP3A4 situated between the LUMO and HOMO orbitals of TCPP (Figure 2B), which offered the possibility of sufficient electron supply from the excited TCPP via Au@CNC to the heme center of enzyme, leading to the enhanced photocurrent.


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In addition, the mass ratio of Au@CNC to TCPP was optimized to enhance the efficiency of the as-prepared bio-nanoreactor. As shown in Figure S4, TCPP exhibited a strong fluorescence peak around 658 nm (curve a). However, by increasing the mass ratio of Au@CNC from 1:10, 1:5, 2: 5 to 4: 5, the FL intensity of TCPP decreased distinctly and then reached a platform (curve b-e). This could be attributed to the covalent binding between Au@CNC and TCPP, and the outstanding conductivity of Au@CNC, which expedited the light-induced electron and energy transfer from the excited TCPP to Au@CNC. Thus, the TCPP/Au@CNC was capable of serving as an excellent light-driven enzymatic reactor, and the final mass ratio of Au@CNC to TCPP was selected as 2:5. 3.3. Light-driven of P450-mediated biocatalysis with Au@CNC bionanoreactor. 7-ethoxytrifluoromethyl coumarin (7-EFC) was chose as a model compound that was able to be metabolized by CYP3A4 to 7-hydroxytrifluoromethyl coumarin (7-HFC) through O-dealkylation reaction. Generally, in the resting state, the heme iron of CYP450 was mainly in low-spin state and had a water molecule at the distal position.6 When the substrate entered the heme pocket and then removed the water molecule, the ferric heme iron equilibrium was shifted to the high-spin form, enabling one electron transfer from CPR to reduce the iron to the ferrous state.43-45 The in vitro CYP3A4-mediated photo-catalytic drug metabolism reaction by CYP3A4/TCPP/Au@CNC was conducted by means of irradiating a mixture of the CYP3A4/TCPP/Au@CNC and 7-EFC under light (0.5 W cm2). As shown in Figure 4A, 7-EFC exhibited a single transition at 382 nm,



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but after the visible light irradiation for 10 min, a new peak appeared at 430 nm, which could be ascribed to the transition of the product (7-HFC). Control experiments showed that no fluorescence characteristic peak of metabolite emerged by using only TCPP or CYP3A4 reacted with the same amount of 7-EFC (Figure 4A). In addition, the coupling of CYP3A4 with TCPP (CYP3A4/TCPP) also displayed some metabolic activity, but the FL intensity of the metabolite was much lower than that of CYP3A4/TCPP/Au@CNC, indicating a low metabolic activity of CYP3A4/TCPP (Figure 4A). When CYP3A4/TCPP was confined into CNC without modification of CNC with AuNPs, the CYP3A4/TCPP/CNC showed a good metabolic behavior, the metabolic activity was higher

than

that

of

CYP3A4/TCPP

but

much

lower

than

that

of

CYP3A4/TCPP/Au@CNC (Figure 4A). Figure 4B showed the corresponding HPLC chromatograms. As illustrated, there was only one peak emerged at the retention time of 6.2 min for pure 7-EFC, while for CYP3A4/TCPP, CYP3A4/TCPP/CNC and CYP3A4/TCPP/Au@CNC systems, a new peak appeared at the retention time of 3.5 min, which could be attributed to the generation of 7-HFC. Similarly, the amount of 7-HFC generated by CYP3A4/TCPP/Au@CNC was much larger than that of CYP3A4/TCPP and CYP3A4/TCPP/CNC, illustrating that a high metabolic activity was achieved by confining CYP3A4/TCPP into the pores of CNC, and the presence of Au NPs enhanced the metabolic activity of CYP3A4/TCPP. All of these results demonstrated that the light-driven drug metabolism could be proceeded more efficiently with Au@CNC as a bionanoreactor, which was capable of providing a pleasant microenvironment to sustain


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the stability and catalytic activity of CYP3A4 and accelerating electron transfer. The time-dependent response showed that the fluorescence intensity of 7-EFC decreased rapidly (about 60%) with the increasing time in 3 min, and then decreased slowly and reached a steady state gradually within 10 min (Figure S5). This indicated that the CYP3A4 mediated O-dealkylation was a fast enzymatic reaction, and 10 min was chosen as the enzymatic reaction time in our following research. The dose-dependent response was investigated by the fluorescent measurements. Due to the emission positions of 7-EFC and 7-HFC were too close that only a partial emission spectrum of 7-HFC could be recorded in the mixture solution of 7-EFC and 7-HFC, Gaussian spectral function was employed to obtain intuitive FL spectrum with two peaks (Figure S7). As shown in Figure 5A, the fluorescence intensity of 7-HFC increased successively with the increasing concentration of 7-EFC. A linear relationship between the concentration of 7-EFC and the FL intensity of the product over a range of 100~3000 nM was obtained (Figure 5B), where the regression equation was I=11.1865c–871.8 (R2=0.9908) with a detection limit of 33 nM at S/N=3. Notably, the apparent Michaelis constant Kmapp was calculated to be 12.83 μM, and the enzymatic rate constant kcat was 13.8 min−1, which was higher than 0.14 min−1 by NH2-PMO−Microsome nanoreactor.30 On account of the inhibition effect of drugs on CYP450 activity was of significance for the development of new drugs and the evaluation of their toxicity, the research of inhibition effect was conducted by adding ketoconazole, a typical and mild inhibitor used for CYP3A4 mediated metabolism, to the mixture of 7-EFC and photochemical



bionanoreactor before the light irradiation. As illustrated in Figure S6, there was no characteristic FL peak of 7-HFC appeared after 10 min visible-light irradiation in the presence of ketoconazole (1.0 μM), suggesting an excellent enzyme inhibition effect of Au@CNC bionanoreactor. Additionally, the specificity of the as-prepared light-driven CYP3A4 mediated in vitro drug metabolism system was also investigated (Figure S8), by using resorufin ethyl ether (7-ER) as a competitive substrate. And the corresponding metabolite resorufin (7-HR) could be monitored with the fluorescence peak at 580 nm.21 Obviously, there was no new fluorescence peak emerged at 580 nm, illustrating the CYP3A4/TCPP/Au@CNC photocatalytic bionanoreactor displayed a favorable substrate specificity. The stability of the CYP3A4/TCPP/Au@CNC bionanoreactor was also examined by periodically measuring the FL intensity of the generated 7-HFC over the storage period of 28 days. The obtained CYP3A4/TCPP/Au@CNC was stored in 0.1 M PB solution at 4°C and keep in dark. As shown in Figure S10, the FL intensity was maintained about 91.1% of its initial response for metabolizing the same amount of substrate (1.0 mM) after 28 days, indicating the CYP3A4/TCPP/Au@CNC bionanoreactor had an acceptable stability. In addition, the CYP3A4/TCPP/Au@CNC showed a remarkable reproducibility for the light-driven metabolic activity and the relative standard deviation (RSD) for six times was 0.85% (Figure S9).

4. CONCLUSIONS


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In summary, a bionanoreactor was fabricated by confining cytochrome P450 enzyme and TCPP inside the pores of Au@CNC, and used for light-driven drug metabolic assay. The deposition of AuNPs in CNC enhanced the conductivity, wettability and biocompatibility of the pristine CNC, which increased the loading amount and stability of enzyme and achieved a high metabolic efficiency. The confined CYP3A4 in Au@CNC exhibited excellent enzymatic activity in terms of fast-response, short assay time (10 min), good selectivity and low detection limit (33 nM). The apparent Michaelis constant Kmapp and the enzymatic rate constant kcat toward substrate of 7-EFC were 12.83 μM and 0.23 s–1, respectively. The linear response of the 7-EFC concentration toward the FL intensity of the product was in range of 100~3000 nM. Notably, the bionanoreactor showed good specificity and remarkable repeatability, which suggested that such a photochemical bionanoreactor had great potentials for the development of new drugs and screening of effective drug candidates.

Associated content Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: xxxxxx. The detailed detection process by HPLC analysis; UV-vis and fluorescence spectrum of TCPP; the XPS spectra and BET tests of CNC and Au@CNC; the fluorescence spectra of different mass ratio of TCPP to Au@CNC; the specificity, reproducibility and stability of the TCPP/Au@CNC bionanoreactor (PDF)



Author information Corresponding Authors *E-mail: [email protected]; [email protected]. Notes The authors declare no competing financial interest. Acknowledgements The authors thank Professor Zheng Hu and Doctor Xizhang Wang from Nanjing University for kindly donate carbon nanocage materials. We gratefully thank the supports from the National Natural Science Foundation of China (grant 21635004, 21627806).


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Figure captions Scheme 1. The fabrication process of the light-driven bionanoreactor assisted by TCPP/Au@CNC for drug metabolism. Figure 1. The SEM and TEM images of CNC (A, C) and Au@CNC (B, D). Insets were the HRTEM images of CNC and Au nanoparticles, respectively. Figure 2. (A) The photocurrent responses of (a) bare, (b) TCPP, (c) TCPP/Au@CNC and (d) CYP3A4/TCPP/Au@CNC modified ITO electrodes in 0.1 M PB (pH 7.4) at a potential of –0.2 V with light on and off, respectively. (B) Schematic illustration of the electron-transfer mechanism for CYP3A4 mediated bioconversion. Figure 3. (A) The UV-vis spectra of CNC, Au@CNC, TCPP/Au@CNC and CYP3A4/TCPP/Au@CNC. (B) Zeta potential of different samples in 0.1 M PB (pH=7.4) at 25 °C. Figure 4. (A) The FL spectra of 7-EFC before and after light irradiation in 0.1 M PB (pH 7.4)

for

10

min

in

the

presence

of

TCPP,

Au@CNC,

CYP3A4/TCPP,

CYP3A4/TCPP/CNC and CYP3A4/TCPP/Au@CNC, respectively. (B) The UV-HPLC chromatograms of 7-EFC, and the metabolic reaction product of 7-EFC under 10 min of the

light-driven

biocatalysis

by

CYP3A4/TCPP,

CYP3A4/TCPP/CNC

and

CYP3A4/TCPP/Au@CNC, respectively. The substrate was observed at tR≈6.2 min and the metabolite of 7-EFC was observed at tR≈3.5 min. Absorbance at 333 nm was recorded.



Figure 5. (A) FL spectra of generated 7-HFC excited at 333 nm in 0.1 M PB (pH 7.4) with different concentrations of substrate metabolized by CYP3A4/TCPP/Au@CNC: (a) 100 nM, (b) 150 nM, (c) 250 nM, (d) 750 nM, (e) 1000 nM, (f) 1250 nM, (g) 1500 nM, (h) 2000 nM, (i) 2500 nM, (j) 3000 nM. (B) The calibration curve of the detection.


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Scheme 1

Scheme 1. The fabrication process of the light-driven bionanoreactor assisted by TCPP/Au@CNC for drug metabolism.



Figure 1

Figure 1. The SEM and TEM images of CNC (A, C) and Au@CNC (B, D). Insets were the HRTEM images of CNC and Au nanoparticles, respectively.


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Figure 2

Figure 2. (A) The photocurrent responses of (a) bare, (b) TCPP, (c) TCPP/Au@CNC and (d) CYP3A4/TCPP/Au@CNC modified ITO electrodes in 0.1 M PB (pH 7.4) at a potential of –0.2 V with light on and off, respectively. (B) Schematic illustration of the electron-transfer mechanism for CYP3A4 mediated bioconversion.



Figure 3

Figure 3. (A) The UV-vis spectra of CNC, Au@CNC, TCPP/Au@CNC and CYP3A4/TCPP/Au@CNC. (B) Zeta potential of different samples in 0.1 M PB (pH=7.4) at 25 °C.


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Figure 4

Figure 4. (A) The FL spectra of 7-EFC before and after light irradiation in 0.1 M PB (pH 7.4)

for

10

min

in

the

presence

of

TCPP,

Au@CNC,

CYP3A4/TCPP,

CYP3A4/TCPP/CNC and CYP3A4/TCPP/Au@CNC, respectively. (B) The UV-HPLC chromatograms of the metabolic reaction product of 7-EFC under 10 min of the light-driven

biocatalysis

by

CYP3A4/TCPP,

CYP3A4/TCPP/CNC

and

CYP3A4/TCPP/Au@CNC, respectively. The substrate was observed at tR≈6.2 min and the metabolite of 7-EFC was observed at tR≈3.5 min. Absorbance at 333 nm was recorded.



Figure 5

Figure 5. (A) FL spectra of generated 7-HFC excited at 333 nm in 0.1 M PB (pH 7.4) with different concentrations of substrate metabolized by CYP3A4/TCPP/Au@CNC: (a) 100 nM, (b) 150 nM, (c) 250 nM, (d) 750 nM, (e) 1000 nM, (f) 1250 nM, (g) 1500 nM, (h) 2000 nM, (i) 2500 nM, (j) 3000 nM. (B) The calibration curve of the detection.


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TOC


Enhanced Metabolic Activity of Cytochrome P450 ... - ACS Publications

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